Enhancement of morphologic detail in tissue

ABSTRACT

Tissue is prepared for microscpic examination by subjecting the tissue to fixation during centrifugation to achieve a significant cell flattening with resultant dispersion of cell contents and improved cytological detail while maintaining the substantial integrity of morphologic pattern of cell entities. A preferred centrifugation apparatus comprises a tissue retaining clip positioned on a centrifuge drum with provision for spraying a programmed sequence of tissue treating substances into said drum.

1 Mar. 11, 1975 ENHANCEMENT OF MORPHOLOGIC DETAIL IN TISSUE [76] Inventor: Kurt W. Mikat, 6061 Rolling Green, Grand Blanc, Mich. 48439 [22] Filed: Dec. 1, 1972 [21] Appl. No; 311,349

[52] U.S. C1. 424/3, 233/26 [51] Int. Cl ..G01nl/00, GOln 1/30, G02b 21/34 [58] Field of Search 424/3; 117/3, 101; 118/44,

3,705,048 12/1972 Staunton 424/3 X OTHER PUBLICATIONS Watson, J. Lab & Clin. Med.,Vo1. 68, Sept. 1966, pp.

494-501. Martinazzi, AJCP, V01. 56, Dec. 1971, pp. 719-722.

Primary Examiner-Albert T. Meyers Assistant ExaminerA. P. Fagelson Armrney, Agent, or Firni-Hauke, Gifford, Patalidis & Dumont [57] ABSTRACT Tissue is prepared for microscpic examination by subjecting the tissue to fixation during centrifugation to achieve a significant cell flattening with resultant dispersion of cell contents and improved cytological detail while maintaining the substantial integrity of morphologic pattern of cell entities. A preferred centrifugation apparatus comprises a tissue retaining clip positioned on a centrifuge drum with provision for spraying a programmed sequence of tissue treating substances into said drum.

7 Claims, 6 Drawing Figures PATENTEUMARI 1 ms suwlq z Fig-'4 BACKGROUND OF THE INVENTION The invention deals with the preparation of human or other tissue for microscopic examination. It has been the usual practice to immerse a sample of tissue successively in a number of tissue processing fluids to fix the tissue, to wash out the fixative, to dehydrate the tissue, to infiltrate the tissue with paraffin, and the like prior to cutting the tissue into sections of desired thickness. The sections are then mounted on slides and stained.

Attempts have been made to enhance cellular detail in such tissue specimens. Some enhancement has been ascribed to a surface tension effect, a flattening by surface tension tending to disperse the cellular components in the plane of the slide surface allowing more discernible cellular detail than if the cell were in its original and generally more spherical shape. Similarly, it has been observed that brief periods of centrifugation of smears; for example, 3 to 30 minutes; result in enhancement of cytological detail with the apparent en largement of the cells as flattening is increased. Similarly, attempts have been made to attain improved results by stretching cells or by applying relatively large pressures to cells.

It is therefore an object of this invention to provide an improved method and apparatus for the enhancement of cellular detail in tissue prepared for microscopic examination.

SUMMARY OF THE INVENTION The present invention provides for the fixation of tissue under centrifugation to achieve flattening and enhanced morphologic detail. The tissue is simultaneously subjected to centrifugation and fixation for a period of time sufficient to achieve the desired flattening of the cells and the desired fixation to prevent the cells from returning to their more natural shapes. By way of example, a period of 16 hours is a suitable period of time.

A preferred centrifuge of the invention comprises a means, preferably a clip and a permeable envelope, for securing a tissue sample to the wall of a centrifuge drum and comprises a means for spraying a fixative or other tissue processing fluid, or a number of fluids sequentially, into the centrifuge drum in a manner to effect the fixing of the tissue while the tissue is being subjected to centrifugation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagrammatic cross sectional view illustrating an embodiment of the invention;

FIG. 2 is an enlarged diagrammatic cross sectional view of the tissue cube shown in FIG. 1;

FIG. 3 is a diagrammatic cross sectional view illustrating the appearance of the tissue cube of FIG. 2 following centrifugation;

FIG. 4 is a diagrammatic sectional view taken along the line 44 of FIG. 3;

FIG. 5 is a diagrammatic cross sectional view of an embodiment of the invention illustrating a preferred manner of centrifugation and fixation of tissue; and

FIG. 6 is an enlarged diagrammatic cross sectional view of the holding clip of FIG. 5 together with a tissue specimen and fluid permeable envelope.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring more specifically to FIG. I. a filter paper disc or sponge disc 10, is placed in the bottom ofa centrifuge cup 12, the disc I0 being wetted with tissue fixing solution such as I0 percent formalin. A disc of smooth paper 14 is placed over disc I0 and a sample oftissue 16 is placed in the center of disc 14. The tissue I6 is covered with a smooth paper disc I8.

Referring more particularly to FIGS. 2, 3. and 4; the tissue cube 16 is shown with embedded spheres. such as sphere 20, which illustrate various cell entities one may desire to view with improved visability. FIG. 2 rep resents the initial fresh tissue, FIG. 3 the flattened tis' sue following centrifugation, and FIG. 4 a section as it would appear on the slide.

Referring more particularly to FIG. 5 a centrifuge, indicated generally as 22, is provided with a housing 24, an access door 26 and a motor 28. The motor 28 drives a shaft 30 which rotates within journal elements 32, 34.

Mounted on shaft 30 for rotation therewith are a processing fluid conduit 36 provided with a plurality of spray nozzles, such as nozzle 38, and a cylindrical centrifuge drum 40 provided on the interior of its outer wall with clips, such as clip 42. Drum 40 is perforated as at 43 and is provided with a bottom drainage port 44.

A cylindrical liner 46 provided with a bottom drainage port 48 is positioned intermediate the drum 40 and housing 24.

A pump 50 selectively draws processing fluid from storage bottles; such as bottles 52, 54, 56; via tubes 58, 60, 62 respectively and via valve 64. Conduit 66 receives said fluid from pump 50 and delivers it to rotating processing fluid conduit 36. Valve 64 is joined to drainage port 48 by tube 65 for the recycling of processing fluid via pump 50 or for discharge via tube 67. A pump 68 pumps a temperature control liquid from a sump pit, indicated generally as 70, through conduit 72 to coil 74 surrounding the liner 46. The coil 74 terminates at opening 76 to return temperature control fluid to sump pit 70.

The clip 42 secures a fluid permeable envelope 78 containing a tissue sample 16 to the centrifuge drum 40 (FIG. 6).

DESCRIPTION OF OPERATION By way of example flat-bottomed, 1 inch diameter centrifuge cups 12 were prepared in the following manner (FIG. 1). A filter paper disc 10 was placed in the bottom of the cup and covered with 1 ml. of 10 percent formalin. A disc of smooth paper 14 slightly smaller than the formalin-soaked disc was placed over the disc 10. Samples of various fresh human and rat tissues including kidney, testis, brain, spleen, liver and striated muscle were cut into cubes l6 measuring approximately 5 mm. per side. Each tissue cube 16 was then placed in the center of a paper disc 14 in a cup 12 and 'covered by a smooth paper disc 18 to concentrate the formalin fumes for fixation.

Using a free-swinging centrifuge head (not shown) on a size 2 International Centrifuge (International Equipment Company, Boston, Mass.) the tissue cubes 16 were centrifuged at 1,000 g (2,200 rev/min) for 16 hr.. They were then removed and processed through paraffin embedding in the usual manner for microscopic evaluation, cutting sections at 2-3 mm thickness (FIG. 4).

Two sets of controls were prepared: One consisted of corresponding tissue cubes fixed in centrifuge cups prepared as for the experimental tissues but without centrifugation. The other set of tissue cubes were fixed and processed as routine surgical specimens by well known prior art methods.

The fixation of tissue under centrifugation, as described, resulted in a pronounced flattening of tissue cubes 16 into disc or pancake forms (FIG. 3). The greatest flattening occurred with the longer periods of centrifugation (16 hr) and with tissues having a loose connective tissue framework as in testis, brain and liver. The more densely fibrous tissues had more resistance to flattening but still showed enhanced detail. The integrity of morphologic patterns of cell entities, that is histological patterns and cellular relationships, were retained in all cases but spread over a large area (F IG. 4).

Ocular micrometer measurements confirmed enlargement of all cellular types, some with doubling of the average diameter. Nuclear details were generally clearer. A few aspects of tissue detail became less clear such as delicate basement membranes, presumably because these elements flattened out in the plane of the slide surface rather than being clearly observed parallel to the path of light. The controls fixed in centrifuge cups appeared similar to those processed as routine surgical specimens with no significant differences in morphology.

The tissue blocks 10 were also rotated 90 and were sectioned across the cells. Definite flattening with apparent elongation of all cells was apparent (FIG. 3).

In a preferred apparatus (FIGS. 5 and 6) for carrying out the simultaneous centrifugation and fixation of tissue, a tissue sample 16 is placed in an envelope 78 which is attached to centrifuge drum 40 by a clip 42 (FIG. 6). The centrifuge drum 40 is then rotated at about 1,000 g, for example, for about 16 hours. The envelope 78 is made of a material which is permeable to the selected fixative or other processing fluid. It, of course, must resist disintegration by fluid action. Generally a porous paper, a loosely woven fabric, or mesh plastic is suitable.

During the centrifugation processing, fluids are drawn sequentially from as many receptacles (e.g., 52, 54, 56) as needed, (for fixatives, wash solutions, dehydration agents, etc.), the sequence and duration of use being controlled by the programmed valve 64. Such programming systems are now well known in the art and their description here would serve no purpose. The processing fluid being pumped by pump 50 at any given time passes through tube 66 which drains into rotating fluid channel 36 surrounding shaft 30. The rotating fluid is projected outwardly through the nozzles 38 to wet the sample 16 and/or to subject the sample 16 to fumes from the projected processing fluid. The processing fluid passes through the openings 43, spins out over the rim of centrifuge drum 40 or drains from the centrifuge drum 40 through port 44 and falls into the bottom of cylindrical liner 46. The processing fluid then drains through port 48 into tube 65 for recyling via valve 64 and pump 50 or, alternatively, for discharge via valve 64 and discharge tube 67.

The temperature of the interior of the centrifuge is controlled by a temperature control fluid which is pumped by pump 68 from sump pit 70 through tube 72 to coil 74. The temperature control fluid returns to the sump pit 70 via discharge tube 76. A temperature control device (not shown) for controlling the temperature of the temperature control device is positioned in sump pit 70.

Although 16 hours of centrifugation and treatment of tissue has been mentioned above as a suitable length of time in which to secure the advantages of the present invention, it is not critical. As indicated one may wish to use a longer period of time in the case of structures particularly resistant to flattening or more slowly responsive to a desirable processing procedure. Similarly, and in the interest of economy, one may find a shorter period of time satisfactory for the purpose at hand. It may be noted, however, that time is here being reckoned in hours whereas in prior art methods the attainment of some improved morphologic detail in smears by centrifugation alone (that is, without simultaneous fixation), time has been reckoned in minutes (3-30 minutes, for example). Similarly, although a centrifugal force of 1,000 g. is suitable, this may be varied to fit specific needs of the investigator in the enhancement of selected detail in a selected tissue.

With further reference to smears, and, for example, to blood smears prepared by spreading the film with the two slide method, cells in the relatively thinner film areas appear relatively larger with their contents more diffuse. In flattened tissue sections prepared in accordance with the present invention the advantage of the relatively larger appearing cells in smears is attained but with a preservation of morphologic cell patterns; that is, the preservation of the spatial and geometric relationship of cell entities in essentially the same pattern as in the unflattened cell with, of course, the exception that such entities are likely to be relatively more dispersed.

Preparation of tissue sections in accordance with the present invention also results in increased cell nuclear diameters, often twice that of control cells a result which is greater than that attained by the mechanical stretching of cells. Where the observed cell is in mitosis the chromosones are more dispersed allowing better evaluation, including counting and classification to a certain extent.

The invention has found use in testicular biopsies for infertility studies where cellular clarity has previously been disappointing, in definitive cellular identification of tumors where lack of cellular differentiation had previously made specific diagnosis difficult, and in tissue chromosonal analysis.

It will be apparent to one skilled in the art to which the invention pertains that various changes and modifications may be made therein without departing from the spirit of the invention or the scope of the appended claims.

I claim:

1. A method for preparing a tissue for microscopic examination said tissue having intercellular connective matter, said method comprising the steps of subjecting said tissue to a centrifugal force of sufficient magnitude and duration to flatten the cells of said tissue and simultaneously treating said tissue with a tissue fixative to prevent, following such centrifugation, said flattened cells from reverting significantly toward their original shape.

2. The method of claim 1 wherein said duration of centrifugation is sufficient to substantially double the cross sectional areaof a cell nucleus.

3. The method of claim 1 wherein said magnitude of centrifugal force and said duration of centrifugation are sufficient to effect a flattening of cells with some dispersion of cell contents but insufficient to destroy morphologic cell patterns.

4. The method of claim 1 wherein said centrifugation step is carried out over a period of about 16 hours.

5. The method of claim 1 wherein said tissue is enveloped in a material which is permeable to tissue processing fluids.

6. The method of claim 1 wherein said tissue fixative is formalin.

7. A method for preparing a tissue for microscopic matter, said method comprising the steps of a. enveloping said tissue in a material which is permeable to a selected tissue fixative;

b. subjecting said tissue to a centrifugal force sufficient to effect a flattening of tissue cells with some dispersion of cell contents but insufficient to destroy morphologic cell patterns;

c. subjecting said tissue, while it is subject to said centrifugal force, to the action of said fixative for 21 period of time sufficient to effect a fixation sufficient to prevent, following such centrifugation, said flattened cells from reverting significantly toward their original shape. 

1. A METHOD FOR PREPARING A TISSUE FOR MICROSCOPIC EXAMINATION SAID TISSUE HAVING INTERCULLAR CONNECTIVE MATTER, SAID METHOD COMPRISING THE STEPS OF SUBJECTING SAID TISSUE TO A CENTRIFUGAL FORCE OF SUFFICIENT MAGNITUDE AND DURATION TO FLATTEN THE CELLS OF SAID TISSUE AND SIMULTANEOUSLY TREATING SAID TISSUE WITH A TISSUE FIXATIVE TO PREVENT, FOLLOWING SUCH CENTRIFUGATION, SAID FLATTENED CELLS FROM REVERTING SIGNIFICANTLY TOWARD THEIR ORIGINAL SHAPE.
 1. A method for preparing a tissue for microscopic examination said tissue having intercellular connective matter, said method comprising the steps of subjecting said tissue to a centrifugal force of sufficient magnitude and duration to flatten the cells of said tissue and simultaneously treating said tissue with a tissue fixative to prevent, following such centrifugation, said flattened cells from reverting significantly toward their original shape.
 2. The method of claim 1 wherein said duration of centrifugation is sufficient to substantially double the cross sectional area of a cell nucleus.
 3. The method of claim 1 wherein said magnitude of centrifugal force and said duration of centrifugation are sufficient to effect a flattening of cells with some dispersion of cell contents but insufficient to destroy morphologic cell patterns.
 4. The method of claim 1 wherein said centrifugation step is carried out over a period of about 16 hours.
 5. The method of claim 1 wherein said tissue is enveloped in a material which is permeable to tissue processing fluids.
 6. The method of claim 1 wherein said tissue fixative is formalin. 